Cdc25A, a dual-specific protein phosphatase, is frequently overexpressed in breast cancer, and associated with poor survival. The long-term goal of this program is to understand the exact role of Cdc25A in breast cancer and establish a scientific basis for anti-cancer therapies targeted on this protein. Cdc25A promotes cell cycle progression by activating cyclin E (A)/Cdk2 during G1 through S, and also cyclin B(A)/Cdk1 at the G2/M boundary. Ectopic expression of Cdc25A shortens the passage through G1 and cooperates with H-ras in transforming mouse embryonic fibroblasts, while silencing of Cdc25A results in arrest at both G1 and G2. We have shown that transforming growth factor-beta (TGFbeta), which is important for morphogenesis and tumor suppression of the mammary gland, downregulates Cdc25A in both transcriptional and posttranslational manners. These observations suggest that Cdc25A could be a critical oncogene in breast cancer, although it has not been demonstrated in vivo using animal models. This proposal is based on our following preliminary data: (i) Cdc25A-heterozygous knockout mice we generated show marked resistance to MMTV-ras induced mammary tumorigenesis;(ii) An MMTV-Cdc25A transgene synergistically cooperates with the MMTV-ras transgene in murine tumorigenesis;(iv) TGF-beta promotes Cdc25A protein degradation in a Smad3-dependent manner and this pathway could be defective in MCF7 and other human breast cancer cell lines;(iii) Cdc25A overexpression inhibits apoptosis signal-regulating kinase-1 (ASK1) and diminishes cellular sensitivity to oxidative stress-induced apoptosis. The central hypothesis evaluated in this proposal is that defective degradation of Cdc25A protein is rate-limiting for initiation and/or progression of breast cancer, causing deregulated control of cell cycle progression and apoptosis. The specific aims are: (1) Determine whether deregulated TGF-beta/Smad signaling is involved in Cdc25A stabilization in human breast cancer cell lines;(2) Determine how reduced Cdc25A expression or altered stability ofCdc25A affects cell cycle progression, cellular sensitivity to oxidative stress, and malignant transformation in response to ras and neu, using MCF-10a cells with Cdc25A siRNA and tetracycline-inducible expression ofCdc25A mutants;(3) Determine how altered Cdc25A expression affects breast tumorigenesis in mice with MMTV-neu, MMTV-myc or MMTV-Wnt-1 transgene, using our novel Cdc25A-knockout and MMTV-Cdc25A transgenic mouse lines. We expect that these studies will provide us with significant insight into the role of Cdc25A stabilization in breast cancer development and a molecular handle to develop effective therapies to target the oncogene.